lnorganic phosphate (Pi) plays an important role in a variety of critical cellular activities. Disorders of extracellular Pi concentration and Pi balance, resulting from an impairment in renal Pi reabsorption are common clinical problems in the general population. Recent studies indicate that alterations in renal cholesterol or glycosphingolipid composition are important mediators in the regulation of renal Pi reabsorption in aging, diabetes mellitus, glucocorticoid excess or deficiency, and alterations in dietary Pi or potassium. The molecular and cellular mechanisms how alterations in cholesterol or glycosphingolipids regulate renal sodium gradient-dependent Pi (Na/Pi) transport however are not known. The specific aims of the current proposal are in a cell culture model, the opossum kidney (Ok) cells, which has many of the physiological and biochemical characteristics of the renal proximal tubule: 1) to determine the acute versus chronic effects of alterations in a) cholesterol or b) glycosphingolipid composition on Na/Pi cotransport activity; 2) to determine if the alterations in cholesterol or glycosphingolipid composition modulate Na/Pi cotransport activity by a) regulating Na/Pi mRNA abundance; or by b) regulating the abundance and the expression of Na/Pi cotransporter protein at the level of the apical brush border membrane; or by c) modulating lipid-protein interactions, including changes in the mobile fraction and lateral diffusion of apical membrane Na/Pi cotransporter proteins.